Figure 1. Serpens mice exhibit diminished T-dependent IgG responses to recombinant Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal). IgG levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The serpens phenotype was identified among G3 mice of the pedigree R0445, some of which showed a diminished T-dependent antibody response to recombinant Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal) (Figure 1). 

Figure 2. Linkage mapping of reduced response to rSFV-β-gal using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 53 mutations (X-axis) identified in the G1 male of pedigree R0488.  Normalized phenotype data are shown for single locus linkage analysis without consideration of G2 dam identity.  Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 53 mutations. The diminished T-dependent antibody response was linked by continuous variable mapping to a mutation in Zeb1:  a T to A transversion at base pair 5,772,455 (v38) on chromosome 18, or base pair 180,596 in the GenBank genomic region NC_000084. Linkage was found with a recessive model of inheritance, wherein 1 variant homozygote departed phenotypically from 5 homozygous reference mice and 10 heterozygous mice with a P value of 8.436 x 10^-6 (Figure 2).  

The mutation corresponds to residue 2,793 in the mRNA sequence NM_011546 within exon 8 of 8 total exons.

The mutated nucleotide is indicated in red.  The mutation results in a cysteine (C) to serine (S) substitution at position 915 (C915S) in the ZEB1 protein, and is strongly predicted by Polyphen-2 to cause loss of function (score = 0.998) (1).

Zeb1 and Zeb2 form the mammalian Zeb family of transcription factors. Zeb1 contains seven C₂H₂-type zinc finger domains, each approximately 23 amino acids in length (2-5) (Figure 3). Four are clustered near the N-terminus (NZF, spanning aa 150-272) and three near the C-terminus (CZF, spanning aa 882-959). Near the center of the protein is a homeobox domain (aa 559-618). Repressor domains have been identified at the N-terminus (aa 19-127) (6), in a large central region between the two zinc finger clusters (aa 303-902) (7), and in two separate regions within aa 303-902 (aa 302-542 and 760-902) (8).  The region between the CZF domain and the C terminus is highly rich in glutamic acid residues (38%), and has been characterized as an activation domain (aa 1011-1124) (9). Three five-amino acid sequences (PLNLC, PLDLS, PLNLS) between the homeodomain and the CZF domain bind to the co-repressor CtBP, and this region (spanning aa 685-749) is designated the CtBP interaction domain (CID) (10). A region between the NZF and homeobox domains of Zeb1 was identified as a Smad interaction domain (aa 377-456) for Smad1, Smad2, and Smad3 (11). Zeb1 synergizes with TGF-β/BMP in transcriptional activation by aiding in the recruitment of p300 and P/CAF (histone acetylases) through a direct interaction involving the N-terminal region of Zeb1 (12). The negative cofactor NC2 binds to amino acids 726-829 of Zeb1 (9).

The serpens mutation results in a cysteine to serine substitution at amino acid 915 within the CZF domain.

Please see the record cellophane for more information about Zeb1.

Mice with a truncation of the C terminus of Zeb1 following amino acid 727 (Zeb1^{ΔC727/ΔC727}), and therefore lacking the cluster of C-terminal zinc fingers and the Glu-rich domain, exhibited lethality within two days after birth (13). Like those of Zeb1^-/- mice, thymi of surviving Zeb1^{ΔC727/ΔC727} mice were smaller and contained 0.2% to 1% the number of thymocytes found in wild type mice (13). The medulla and cortex were indistinguishable upon histological analysis of Zeb1^{ΔC727/ΔC727} thymus sections. Spleen size and cellularity were similar between Zeb1^{ΔC727/ΔC727} and wild type mice, although there was a trend toward reduced cellularity in Zeb1^{ΔC727/ΔC727} mice.  Lymph node cellularity was 10% of that in wild type mice. The ENU-induced mutant mice, termed cellophane, exhibited reduced B cell proliferation in response to BCR crosslinking (14). The reduced ability of cellophane B cells to proliferate in response to BCR stimulation is proposed to lead to impaired antibody responses and germinal center formation following immunization. Similar to cellophane mice, the serpens mice also exhibited impaired antibody responses to the T-dependent antigen, indicating reduced function of Zeb1^serpens.

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold

The following sequence of 472 nucleotides is amplified (chromosome 18, + strand):

1   tggcctcatc tgcaaagcag tgtataacct tgaattttca catagaaaaa aatgaaccat
61  agtattatcg tacaattggg aacctgccag tttcttgacc atggaactgt gaccctcccc
121 ttgcacaatc tgaatgtccc ttcattctcc caagcactct gtttatttct gccagtgttc
181 tgtttcatag ctgcgttctt accttgcagg taagaggcct cacgagtgtg gaatctgtag
241 aaaggcattt aaacacaagc atcatttgat tgagcacatg cggctgcact ctggggaaaa
301 gccctatcaa tgtgacaagt gtggcaagcg cttctcacac tccggctcct actctcaaca
361 catgaatcac cgctactcct actgcaagag aggagctgaa gacagagatg ctatggagca
421 ggaagacgct gggcccgaag tcctgccgga agtcctggcg actgagcatg tt

Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.